PROJECT SUMMARY The past decade has witnessed dramatic improvements to cryo-electron microscopy (cryo-EM) instrumentation, making it possible to image the ultrastructure of a cell of interest using cryo-electron tomography (cryo-ET). Platelets play a vital role in hemostasis by forming a clot and stopping bleeding at the site of vascular injury. Resting platelets are discoid in shape. Upon activation platelets undergo dramatic morphological changes, including cytoskeletal rearrangement, membrane receptor activation and redistribution in the plasma membrane, and granule release. These structural changes are linked to platelet dysfunction and have implications for bleeding disorders including platelet storage lesions, thrombocytopenia, and thrombosis. Current understanding of platelet ultrastructure is derived mainly from transmission electron microscopy (TEM) studies in the 1950-1980?s. Conventional chemical fixation of biological samples for TEM is known to cause structural artifacts in platelet organelles and macromolecules. The sizes of platelets, 3-5 m in diameter and 1-2 in thickness, is conducive to whole cellular cryo-ET, we propose in this project to apply cutting-edge cryo-ET methods to platelets and develop effective workflows in two Specific Aims. In Specific Aim 1, we will develop multimodal protocols for 3- dimensional (3D) cryo-ET imaging of human and murine platelets. New imaging methods including whole cellular cryo-ET, correlative light electron microscopy, and hole-free phase-plate contrast enhanced cryo- ET will be developed to visualize platelets from wild-type mice, healthy human donors, and patients with abnormal granules. Protocol development includes cryo-specimen preparation, image acquisition, image analysis with an emphasis on visualization and quantification of platelet organelles and macromolecules. In Specific Aim 2, we will apply the developed cryo-ET protocols to characterize the 3D ultrastructure of platelets with therapeutic implications. As short shelf-life of stored platelets contributes to the severe shortage of platelets available for transfusion treatment in the hospital, refrigeration is a potentially promising method to store platelets in order to minimize bacterial growth and reduce metabolism during storage. However, refrigeration causes morphological changes of platelets and leads to their fast clearance after transfusion. We will use refrigerated platelets as a model system for developing the cryo-ET imaging protocols. Characterization and comparison of cellular ultrastructure in fresh and refrigerated platelets will be carried out, with a focus on changes in microtubules and the actin cytoskeleton, and clustering of platelet receptors on the plasma membrane. Overall, in this project we propose to establish a robust, cutting-edge cryo-ET imaging protocol for platelets, which can be adapted to other types of cells. Visualization of platelets at unprecedented structural resolution will also enable detailed comparison of healthy and diseased platelets, establishing a productive platform for studying platelet physiology and pathophysiology.